Nociceptin/orphanin FQ (N/OFQ), via its receptor NOP, modulates nociception, stress, and anxiety. N/OFQ is regarded as an anti-opiate peptide because central N/OFQ causes pronociception and reversal of stress- induced analgesia (SIA). However, N/OFQ can affect nonopioid-mediated analgesia, and its anxiolytic effect is unlikely to be mediated through interaction with the opiate system. Both N/OFQ and NOP are abundant in the hypothalamus, where hypocretins/orexins (Hcrts) are selectively synthesized. The Hcrts regulate wakefulness and alertness, modulate nociceptive processing, and contribute to SIA. Because N/OFQ and Hcrts produce opposite modulations for most behaviors and cellular actions assessed, we hypothesize that N/OFQ exerts its effects on neurobehavior, primarily SIA and fear or stress-induced anxiety-like behavior, through direct modulation of Hcrt neuronal activity in the lateral hypothalamus (LH) and/or interaction at the projected sites that receive both N/OFQ and Hcrt inputs and co-express their cognate receptors. Our preliminary studies show that N/OFQ-containing fibers contact Hcrt neurons and that N/OFQ inhibits Hcrt neuronal activity via both pre- and postsynaptic mechanisms. N/OFQ also depresses cytoplasmic Ca2+ in a majority of the Hcrt neurons in orexin/cameleon 2.1 mouse hypothalamic slices. We found that orexin/ataxin-3 neurodegenerative (orexin/ataxin-3) mice exhibit no SIA in the restraint model. Conversely, intracerebroventricular (icv) administration of Hcrt mimicked SIA in orexin/ataxin-3 mice animals but prevented SIA in wildtype (WT) mice. Furthermore, exogenous Hcrt restored SIA in animals treated with centrally administered N/OFQ. These preliminary results support our hypothesis and lead us to propose the following Specific Aims: 1. Test the hypothesis that N/OFQ-containing fibers contact Hcrt neurons and determine the anatomical source(s) of N/OFQ innervation by using standard anatomical techniques (e.g., immunohistochemistry, in situ hybridization, and retrograde tracing). We will use using multiple labeling quantitative electron microscopical (EM) techniques to further determine whether N/OFQ-containing fibers synaptically contact Hcrt neurons. 2. Characterize the cellular physiological modulation of N/OFQ on Hcrt neurons by using patch clamp recordings of Hcrt neurons from orexin/EGFP mice and by Ca2+ imaging using orexin/cameleon 2.1 mice. 3. Test the hypothesis that N/OFQ blocks SIA partly through the modulation of the Hcrt system by investigating N/OFQ effects on nociceptive processing in WT and orexin/ataxin-3 mice. 4. Test the hypothesis that N/OFQ exerts anxiolytic-like effects partly via inhibition of the Hcrt-mediated stress and anxiety responses in mice using both conditioned and unconditioned fear paradigms. The proposed research will reveal whether there is an integrated neuronal circuit linking the N/OFQ and Hcrt systems that provides a dual-modulation to balance stress responses particularly related to nociceptive processing and stress adaptation.